New therapies that protect or restore bone quality have great potential to significantly improve skeletal health. Development of these therapies requires the identification of the cellular and molecular mechanisms that regulate bone quality. Therefore, the long-term goal of this research is to identify mechanisms by which biological and physical cues control bone extracellular matrix (ECM) material properties. To that end, this renewal application builds on our discovery that TGF regulates the material properties of bone ECM and remains one of just a handful of factors yet shown to do so. Recent work, supported by the first funding cycle, establishes osteocytes and MMP13 as key participants in the control of bone quality through a dynamic process called perilacunar remodeling (PLR). In PLR, osteocytes secrete proteases such as MMP13 to resorb the perilacunar bone matrix. PLR is essential for the maintenance of bone quality, systemic mineral homeostasis, and the canalicular channels that facilitate osteocyte nourishment, communication, and mechanosensation. Our recent data suggests that disruption of PLR in human bone may contribute to the bone fragility in diseases such as osteonecrosis of the femoral head or osteoradionecrosis of the jaw. However, major gaps surround the understanding of the role or regulation of PLR in healthy bone or in skeletal disease. Preliminary data raise the possibility that PLR is regulated by mechanical load and by TGF, a mechanism that has the potential to couple the maintenance of bone quality by PLR to changing physical and biological demands on the skeleton. This proposal tests the hypothesis that osteocytes regulate bone ECM material properties through perilacunar remodeling in a load- and TGF-dependent manner to control bone quality and cellular tension. Specifically, this project aims to: 1) determine the extent to which perilacunar remodeling is mechanosensitive and TGF-regulated, 2) identify mechanisms by which mechanical load regulates the effects of TGF on bone, and 3) determine the functional impact of PLR on bone ECM quality and osteocyte tension. PLR activity and regulation will be evaluated using a combination of histologic, radiologic, bioengineering, and molecular approaches. These studies will assess the effects on PLR of osteocyte-specific MMP13-deficiency, applied mechanical loads, and pharmacologic inhibition of the TGF type I receptor. In vivo and in vitro gain and loss of function studies will be used to determine the extent to which these factors operate in an epistatic pathway. In addition, this project examines the effect of dynamically regulated perilacunar bone ECM material properties on osteocyte cellular tension, signaling, and function. This project is significant because it will elucidate the regulation of osteocyte mediated PLR and bone quality by TGF and physical cues, revealing new mechanisms that can be therapeutically targeted to prevent bone fragility and maintain skeletal health.